Fluorescent probes abound in cellular, molecular, and even whole-animal biological research today. Indeed, so many fluorescent probes are available to researchers – why and how should we develop new ones? “We like to start with the question of biological need,” says Marcel Bruchez, associate professor of chemistry and biological sciences at Carnegie Mellon University. “What do we need to see that we cannot see with existing probes?” Researchers can meet this need by creating a new fluorescent probe or improving existing probes.
Once you've identified a need for a new or improved probe, the detailed processes of design and optimization begin. According to Bruchez, developing a new probe is “half rational design, half ruthless opportunism.”
From quantum dots to probes
Prior to developing fluorescent probes, Bruchez pioneered quantum dots, which are tiny crystals used to fluorescently label proteins or cells. To commercialize this technology, he founded the Quantum Dot Corporation in 1998, which was later acquired by Invitrogen in 2005. At Quantum Dot, Bruchez fielded a lot of support calls, which he actually enjoyed. “Every support call was a potential scientific project,” he recalls. “I established many collaborations that I still enjoy today.”
But he also learned that this was not best for business. “This experience taught me the value of offering a finished product, something that promises to do a particular thing, and does it robustly all the time,” he says. Today Bruchez bears this in mind with his new company, Sharpe Edge Labs, which he co-founded with Carnegie Mellon colleague Alan Waggoner to sell assays for receptor trafficking and channels.
Although quantum dot technology is still being developed, Bruchez is no longer involved. “I knew too much about the proprietary materials to continue to improve and elaborate on that particular chemistry,” he says, “and there were literally dozens of groups working on alternative surface chemistry approaches to address the known shortcomings of the quantum dots – size, valency, nonspecific binding, inconsistency, and some would say blinking. So it was not clear at the time how unsolved the issues would remain today.”
Instead, Bruchez was drawn to uncharted territories and the exacting nature of molecular design. For example, his recent work includes a pH sensor probe developed to facilitate labeling the fraction of G-protein-coupled receptor (GPCR) proteins expressed on the surfaces of live cells’ membranes (1). Previous methods involved allowing labeled membrane proteins to be taken up by endocytosis, then stripping all other proteins off the cell surfaces with trypsin.
In contrast, Bruchez’s group developed a faster and more convenient color-change assay based on intramolecular energy transfer. The assay makes receptors on the cell surface fluoresce one color, while internalized receptors within endosomal vesicles fluoresce a different color.
“Physiologically, endocytosed receptors undergo changes in pH, so we reasoned that a pH sensor could give us data about surface and endosomal fractions, and also some information about average pH of the endocytosed pool,” says Bruchez. “The dye bound at the surface, with an ‘inactive’ pH sensitive chromophore, [emits] high fluorescence from the bound fluorogen in green. Upon internalization, the pH dependent dye is activated, quenching the fluorescence of the fluorogen, and changing the emitted color to red, with the same excitation wavelength.”
A little help from libraries
Meanwhile, other labs are taking different approaches to develop fluorescent probes. For example, Young-Tae Chang, head of the Laboratory of Bioimaging Probe Development at the Singapore Bioimaging Consortium, uses a method called diversity-oriented fluorescence library approach (DOFLA). The technique lets researchers zero in on probes using a library of small, structurally-diverse fluorescent compounds.
“This is especially useful when the target or biomarker is unclear,” says Chang. “For example, to make a probe selective for stem cells without a rich knowledge base concerning the cell type, we can do image-based screening of the stem cells using the library of fluorescent compounds. So far, we have made more than 10,000 compounds, and in our experience, finding selective probes from this type of screening is almost 100% successful.”
Recently Chang’s group used DOFLA to create fluorescent probes for embryonic and neuronal stem cells. Chang envisions these tools supplanting current antibody-based methods for the detection, isolation, or reprogramming of stem cells from primary tissues. The DOFLA method may not be accessible to everyone, as it does require an already well-established screening system of pure or enriched stem cell cultures. However, its power holds therapeutic promise.
“A cancer stem cell probe will be an important next step, and reliable culture conditions without diluting the population would be the key,” says Change. “Once visualized, they can be killed photodynamically. This is our next move.”
In another library-based approach, Armitage’s group is using synthesized fluorogenic dyes to select proteins from a combinatorial library that will bind to the dyes and also activate their fluorescence. They term the complex of dye+protein a fluoromodule, which “then becomes a potential GFP-like tag that can be applied to studying protein localization and function,” says Armitage. Recently they developed a dye-protein fluoromodule that could be excited at 405 nm, as well as protein that bound to not one but several dyes, such that it covered nearly the entire visible spectrum.
Armitage’s group is also assembling scores of intercalating dyes on DNA nanostructure scaffolds. “The resulting DNA-dye complexes are exceptionally bright, fluorescent labels that we are now using for cell surface imaging, and in vitro protein detection,” he says. “The rationale for this approach is that the brighter fluorescence offered by the DNA ‘nanotag’ should permit more sensitive detection of the biomolecular target.”
A fluorescent future
Bruchez expects that future fluorescent probe development will focus on tools that can tag genetically-encoded proteins inside cells in multiple colors, with dyes suitable for the advanced measurements that electrophysiology, super-resolution imaging, and protein-protein interactions can entail. "You can put new probes directly into the endogenous genetic locus," says Bruchez. "This allows you to put your fluorescent probe directly onto the copy of the protein that the cell would normally make, rather than adding a new copy on an expressible plasmid."
A major bottleneck in labeling, however, is the large resources required for generating stably-transfected cell culture lines or transgenic animals. One still sought-after solution is the ability to label endogenous proteins. “Approaches like aptamer molecular beacons and fluorogenic aptamer switches are showing some promise in this direction, but new approaches will be needed to generalize this,” says Bruchez. “These sensors must be bright, specific, stable, and capable of getting into living cells without harsh treatments.”
Armitage agrees that in efforts to avoid over-expression, it will be difficult to obtain alternative fluorescent probes with sufficient selectivity, affinity, brightness, and photostability, but it seems there is hope of a hard-earned success. “Technologies that combine intracellular expression and labeling by exogenous dyes either by covalent or noncovalent binding are promising,” Armitage says, “but there is a great deal of work still to be done.”
1. Grover A, Schmidt BF, Salter RD, Watkins SC, Waggoner AS, Bruchez MP. (2012) Genetically encoded pH sensor for tracking surface proteins through endocytosis. Angew Chem Int Ed Engl. 51(20):4838-42.